Regulatory network for FOREVER YOUNG FLOWER-like genes in regulating Arabidopsis flower senescence and abscission

FOREVER YOUNG FLOWER (FYF) has been reported to play an important role in regulating flower senescence/abscission. Here, we functionally analyzed five Arabidopsis FYF-like genes, two in the FYF subgroup (FYL1/AGL71 and FYL2/AGL72) and three in the SOC1 subgroup (SOC1/AGL20, AGL19, and AGL14/XAL2), and showed their involvement in the regulation of flower senescence and/or abscission. We demonstrated that in FYF subgroup, FYF has both functions in suppressing flower senescence and abscission, FYL1 only suppresses flower abscission and FYL2 has been converted as an activator to promote flower senescence. In SOC1 subgroup, AGL19/AGL14/SOC1 have only one function in suppressing flower senescence. We also found that FYF-like proteins can form heterotetrameric complexes with different combinations of A/E functional proteins (such as AGL6 and SEP1) and AGL15/18-like proteins to perform their functions. These findings greatly expand the current knowledge behind the multifunctional evolution of FYF-like genes and uncover their regulatory network in plants.

W e have previously reported that Arabidopsis FOREVER YOUNG FLOWER (FYF) specifically regulates flower organ senescence and abscission by suppressing the downstream genes of ethylene signaling EDF1/2/3/4 and abscission-associated genes BOP1/2 and IDA 1,2 . Conserved functions in regulating flower organ senescence and abscission have been reported for FYF orthologs identified in different species of orchids [3][4][5] . Based on sequence homology and phylogenetic analysis, six putative closely related FYF-like genes could be subjected to two subgroups: (1) the FYF group composed of FYF, AGL71, and AGL72 and (2) the SOC1 group composed of AGL20 (SOC1), AGL19, and AGL14 (XAL2) in the Arabidopsis genome 6 .
It has been reported that additional new genes could be generated in the genome through gene duplication, which was thought to play an important role in organisms during evolution [7][8][9] . Phylogenetic analysis revealed that three duplication events from an FYF-like ancestor may have occurred (two within subgroups) to generate six Arabidopsis FYF-like genes 6 . Functional analysis indicated that the majority of the duplicate FYFlike gene pairs (FYF/AGL71/AGL72/SOC1/AGL19/AGL14) may retain overlap of the original ancestral function, such as the regulation of flowering time [10][11][12][13][14][15][16][17][18][19][20][21][22][23] , or have specific subsets of the original ancestral function, such as the regulation of root 24,25 and ovule 26 development as seen for AGL14 (XAL2), which was described as subfunctionalization 9,[27][28][29][30] . Although different putative functions were uncovered for these Arabidopsis FYF-like genes, exploration of the function in regulating flower organ senescence and abscission was investigated for only the FYF gene and has never been reported for other five Arabidopsis FYF-like genes. It is therefore not clear whether the FYF gene evolved to have this unique function in regulating flower organ senescence and abscission from its ancestor or whether some of the other FYF-like genes also harbored this function during evolution. To uncover these questions, we comprehensively functionally characterized all putative Arabidopsis FYF-like genes for their involvement in regulating flower organ senescence and abscission. Furthermore, we used a FRET-based strategy to investigate the possible heterotetrameric protein complexes formed by the interactions of FYF-like and other MADS box proteins to further verify the regulatory networks for these duplicate FYF-like gene pairs in Arabidopsis.
Here, we show that all other five Arabidopsis FYF-like genes have a function in regulating flower senescence and/or abscission similar to FYF. In this work, we also found that FYF-like proteins can interact with different combinations of A/E functional proteins (such as AGL6 and SEP1) and AGL15/18-like proteins (such as AGL15/18) to form heterotetrameric complexes in regulating flower senescence and abscission.
The distinct expression patterns of FYL1 and FYL2. To investigate the expression patterns of the FYL1 and FYL2 genes, FYL1/2 expression was further analyzed in flowers at different developmental stages. The results indicated that higher FYL1/2 expression was observed during early flower development (before stage 9) than during late developmental stages (after stage 12) ( Supplementary Fig. 3a, b), which was similar to the spatial and temporal expression pattern of FYF during flower development ( Supplementary Fig. 3c) 1 . When FYL1::GUS and FYL2::GUS constructs were generated and transformed into Arabidopsis, GUS staining was exclusively detected in the abscission zone (AZ) of the sepals/petals of FYL1::GUS flowers (Fig. 1a-c) and shows a more extended pattern in the sepals/petals of FYL2::GUS flowers (Fig. 2a-d). Further RT-qPCR analysis indicated that FYL1 expression was highly detected whereas FYL2 expression was almost undetectable in the AZ (Supplementary Fig. 3d). This result is interesting since GUS staining was detected in both sepals/petals and in the abscission zones of FYF::GUS flowers 1 , suggesting that FYL1 and FYL2 might have different subfunctions of FYF in regulating flower senescence and abscission. Furthermore, enhancement of the delay of flower senescence/abscission was observed in 35S::FYL1+SRDX transgenic plants (Fig. 1d, middle and 1f), and an opposite promotion of flower senescence/ abscission was produced in 35S::FYL1-DR+VP16 transgenic plants (Fig. 1d, bottom left and 1g), suggesting that FYL1 should act as a repressor in suppressing flower senescence/abscission, similar to FYF. Once it has been converted into an activator, FYL1-DR+VP16, an opposite dominant negative mutant phenotype will be observed. We also found that the expression of the senescence-associated gene SAG12, downstream genes in ethylene signaling EDF1-3, and ERF1 and abscission-associated genes BOP1/2, IDA, and HAESA were all downregulated in 35S::FYL1 and 35S::FYL1+SRDX plants ( Fig. 1h-j). In addition, 35S::FYL1 and 35S::FYL1+SRDX flowers were insensitive to ethylene treatment (Fig. 1k). Our data suggest that FYL1 could have a prominent role like FYF in controlling floral senescence/abscission once ectopically expressed in Arabidopsis flowers. However, FYL1 should only have a partial role in controlling floral abscission in real life since its expression was restricted to the abscission zone (AZ) of the sepals/petals of flowers (Fig. 1a-c and Supplementary  Fig. 3d).
To further confirm the relationship between FYL1 and sepal/ petal abscission, the FYL1 gene driven by the IDA promoter (IDA::FYL1) was transformed into Arabidopsis. A clear delay in the abscission of the perianth organs was observed in IDA::FYL1 flowers (Fig. 1l-n). However, senescence of the perianth normally occurred from positions 4-5 in these flowers that were not abscised in the IDA:FYL1 transgenic plants (Fig. 1l-n). This phenotype was very similar to what has been observed in ida mutants 31 and IDA::FYF plants 1 ; thus, the result supported the role of FYL1 as a suppressor and its function together with FYF in suppressing IDA and sepal/petal abscission. Arabidopsis were generated. 35S::FYL2 plants surprisingly showed promotion of both flower senescence and abscission (Fig. 2e, first row and 2f). A similar promotion of flower senescence/abscission was observed in 35S::FYL2-DR+VP16 transgenic plants (Fig. 2e, third row and 2h), and an opposite delay of flower senescence/ abscission was produced in 35S::FYL2+SRDX transgenic plants (Fig. 2e, second row and 2g), suggesting that FYL2 should act as an activator in promoting flower senescence/abscission, in contrast to FYF and FYL1. We also found that the expression of EDF1-4, ERF1, BOP1/2, IDA, and HAESA were all downregulated in 35S::FYL2+SRDX plants ( Fig. 2i-k). By contrast, SAG12, EDF1-4, ERF1, IDA, and HAESA were all upregulated in 35S::FYL2 and 35S::FYL2-DR+VP16 transgenic plants (Supplementary Fig. 4). In addition, 35S::FYL2+SRDX flowers were COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03629-w ARTICLE COMMUNICATIONS BIOLOGY | (2022) 5:662 | https://doi.org/10.1038/s42003-022-03629-w | www.nature.com/commsbio insensitive to ethylene treatment (Fig. 2l). This result suggested that FYL2 should function opposite to FYF and could have the same role as FYF once it is converted into a repressor as FYL2+SRDX, and a dominant negative mutant phenotype in suppressing floral senescence/abscission will be observed. However, FYL2 should only have an opposite role to FYF in controlling floral senescence in real life since its expression was specifically in the sepals/petals of flowers ( Fig. 2a-d).
To further confirm the relationship between FYL2 and FYF in regulating sepal/petal senescence, 35S::FYF and 35S::FYL2 were doubly transformed into Arabidopsis, and plants ectopically expressing FYF and FYL2 were generated simultaneously. A clear wild-type-like phenotype by senescence and abscission of the perianth organs at approximately positions 3-4 was observed in the 35S::FYF/35S::FYL2 flowers (Fig. 2m, right), which was earlier than that in the 35S::FYF flowers (Fig. 2m, left) and later than that in the 35S::FYL2 flowers (Fig. 2e, first row). This 35S::FYF/ 35S::FYL2 intermediate phenotype between 35S::FYF and 35S::FYL2 clearly indicated an antagonistic relationship between FYF and FYL2. Thus, the results supported a role for FYL2 as an activator with a function antagonistic to part of the FYF function in controlling senescence of the sepal/petal. FYF can interact with AGL6 and SEP1 in regulating flower abscission/senescence. To investigate which proteins could possibly interact with FYF to form a complex in regulating flower organ abscission and senescence, two potential candidates, AGL6 and SEP1, which have been reported to be able to interact with FYF through yeast two-hybrid screen 32 , were identified. It is important to determine whether the spatial and temporal expression patterns of the AGL6 and SEP1 genes were correlated with FYF. Based on the AGL6::GUS assay, AGL6 expression could be detected in the basal parts and abscission zone of the floral organs during floral development ( Supplementary Fig. 5a-c) 33 and was detected to be more abundant in wild-type flowers before (BP) than after (AP) pollination ( Supplementary Fig. 5d), which overlapped with the expression pattern of FYF. SEP1 has been reported to be expressed in all four whorls of flower organs, is more abundant during early morphological differentiation than in mature flowers, and was not reported in the abscission zone during floral development 34,35 . These results suggested that FYF might interact with AGL6 to form a complex in regulating sepal/ petal abscission. In addition, FYF can interact separately with SEP1 and AGL6 to form a complex in regulating sepal/petal senescence.
We further performed FRET analyses by using FYF-CFP and AGL6/SEP1-YFP to observe physical interactions of FYF and AGL6/SEP1 protein complexes in tobacco cells 36 . The results confirmed that FYF/AGL6 formed heterodimers with high efficiency in the nucleus (Fig. 2n, column 1, Fig. 3a). The same result was observed for FYF/SEP1 (Fig. 2o, column 1). These results suggested that FYF is able to interact with AGL6 and SEP1 to form complexes in regulating flower senescence and/or abscission.
FYL1 can interact with AGL6 in regulating flower abscission. To further investigate whether FYL1 could also interact with proteins similar to FYF in regulating flower organ abscission, FRET analyses were performed. When FYL1-CFP and AGL6/ SEP1-YFP were used to observe the physical interactions of FYL1 and AGL6/SEP1, FYL1/AGL6 can form heterodimers with similar efficiency to FYF/AGL6 in the nucleus of tobacco cells (Fig. 2n, column 2, Fig. 3b). However, the efficiency for the formation of FYL1/SEP1 (Fig. 2o, column 2) was clearly lower than that for FYF/SEP1 (Fig. 2o, column 1). These results suggested that FYL1 is able to interact with AGL6 in a more stable manner than with SEP1 to form complexes. Since FYL1 was only expressed in the AZ of the sepals/petals and could only regulate flower organ abscission, it is reasonable to believe that FYL1 can only physically interact with AGL6 in the AZ to regulate abscission of the sepals/petals. Although they can interact, the FYL1/SEP1 complex should not exist during Arabidopsis flower development since these two genes have no overlapping expression pattern.
The ability of AGL6 to interact with FYF and FYL1 to form complexes reveals that its function should be related to FYF/ FYL1. This assumption was further supported by the result that a similar delay in the flower senescence/abscission phenotype (Supplementary Fig. 5e-i) and downregulation of EDF1, BOP1/2, IDA, and HAESA ( Supplementary Fig. 5j, k) were observed in 35S::AGL6+SRDX plants. This result revealed that FYF/FYL1 can interact with AGL6 to target similar downstream genes once expressed in the same places.
FYF and FYL1 can interact with AGL6/AGL6 and AGL15 proteins to form stable heterotetrameric abscission complexes. Based on the floral quartet model in which plant MADS-box proteins function as higher-order tetrameric complexes 37 , we hypothesized that FYF-AGL6 heterodimer proteins would further form heterotetrameric complexes with other MADS box proteins in the AZ to regulate sepal/petal abscission. It is interesting to note that two MADS box genes, AGL15 and AGL18, have been reported to be expressed in flower organs and in the AZ of  (i) and BOP1/2, IDA and HAESA (j) expression in 35S::FYL1 and 35S::FYL1+SRDX Arabidopsis. Error bars show ± SD. n = 3 biologically independent samples. The expression of each gene in the transgenic plants is given relative to that of the wild-type plant, which was set at 1. The letter "a", "b" and "c" indicates significant difference from the wild-type (WT) value (a: P < 0.05, b: P < 0.01, and c: P < 0.001). The two-sided Student's t-test was used. k Flowers along the inflorescence of 35S::FYL1 (first row), 35S::FYL1 + SRDX (second row), and wild-type (third row) plants after exposure to ethylene. Bar = 2 mm. Inflorescence of IDA::FYL1 plants (l) and flowers along the inflorescence (m) of IDA::FYL1 (bottom) and wild-type (WT, top) plants. The flower organs (arrowed) remained on the IDA::FYL1 flower and siliques. Bars = 2 mm. n Magnified view of an IDA::FYL1 flower with a senescent but not abscised phenotype from (m). s: sepal, p: petal, st: stamen. Bar = 0.5 mm.
supports the notion that AGL15/AGL18 function similarly to FYF/FYL1 as repressors 39 in regulating flower organ abscission. To further explore whether AGL15/AGL18 could also interact with proteins similar to FYF/FYL1 in regulating flower organ abscission, FRET analyses were performed. The results indicated that AGL15/AGL6 are able to form heterodimers with similar efficiency to FYF/AGL6 in the nucleus of tobacco cells (Fig. 2n,   column 6, Fig. 3c). In contrast, AGL18 showed a very weak interaction with AGL6 (Fig. 2n, column 7). This result revealed that AGL15 can form a complex with AGL6, whereas AGL18 might form different protein complexes to perform the redundant function in regulating flower organ abscission.
FYL2 can interact with AGL6 and SEP1 in regulating flower senescence. Similarly, the investigation of whether FYL2 could also interact with proteins similar to FYF in regulating flower organ senescence was also performed using FRET analyses. When FYL2-CFP and AGL6-YFP or SEP1-YFP were used to observe the physical interactions of FYL2 and AGL6 or SEP1, a lower efficiency of FYL2/AGL6 heterodimer formation than that of FYF/ AGL6 (Fig. 2n, column 1) in the nucleus of tobacco cells was observed (Fig. 2n, column 3). The efficiency for the formation of FYL2/SEP1 (Fig. 2o, column 3) was also lower than that for FYF/ SEP1 (Fig. 2o, column 1). These results suggested that similar to FYF, FYL2 can also interact with AGL6 and SEP1 to form senescence complexes, although at a lower efficiency. Since FYL2 was only expressed in the flower organs of sepals/petals, which overlapped with part of AGL6 and SEP1 expression, these data revealed that FYL2 can physically interact with AGL6 and SEP1 during Arabidopsis flower development to regulate sepal/petal senescence.
Since we have already shown that FYL2 functions opposite to FYF in controlling sepal/petal senescence, FYL2 might compete to bind the interacting protein to form a functional complex. To examine this assumption, FRET efficiency for the formation of FYF-CFP/SEP1-YFP complexes was examined in tobacco cells by adding different amounts of unlabeled FYL2 proteins. The results indicated that the efficiency for FYF-CFP to interact with SEP1-YFP (Fig. 2p, column 1) was clearly decreased by the presence of 25-75% of the FYL2 proteins (Fig. 2p, columns 2 and 3). The ability of FYF-CFP to interact with SEP1-YFP was almost completely competed for by the presence of 100% FYL2 protein (Fig. 2p, column 4). Thus, FYL2 competes with FYF to interact with SEP1, performing opposite functions in controlling sepal/ petal senescence.
FYF-like genes AGL19/14 and SOC1 are complementary to FYF in regulating flower senescence. We found a possible mechanism involving three FYF-like genes (FYF and FYL1/2) in regulating flower organ senescence and abscission. It is interesting to note that three other genes in the SOC1 subgroup, AGL19, AGL14 (XAL2), and AGL20 (SOC1), were also closely related to the FYF/ FYL1/FYL2 genes ( Supplementary Figs. 1, 2) 6,16 . Do these three genes also harbor similar functions to FYF/FYL1/FYL2 in regulating flower senescence/abscission? Interestingly, similar to that observed in 35S::FYF and 35S::FYF+SRDX Arabidopsis, a strong delay in flower senescence and abscission (Fig. 4a-c), insensitivity to ethylene treatment (Fig. 4d-i) and downregulation of EDF1-4, ERF1, BOP2, IDA, and HAESA (Fig. 4j, k) were observed in 35S::AGL19 and 35S::AGL19+SRDX Arabidopsis. In contrast to AGL19, only 35S::AGL14+SRDX and 35S::SOC1+SRDX caused a strong delay in flower senescence/abscission and a downregulation of senescence/abscission-related genes ( Supplementary  Figs. 7a-d, 8a-c), whereas no or a reduced effect was seen in 35S::AGL14 and 35S::SOC1 plants. These results indicated that AGL19 and AGL14/SOC1 functioned as strong and weak repressors, respectively, and that part of their function The expression of each gene in the transgenic plants is given relative to that of the wild-type plant, which was set at 1. The letter "a", "b" and "c" indicates significant difference from the wild-type (WT) value (a: P < 0.05, b: P < 0.01, and c: P < 0.001). The two-sided Student's t-test was used. l complemented FYF in suppressing flower organ senescence/ abscission.
Similar to the expression pattern of FYF/FYL2, higher AGL19/ AGL14/SOC1 expression was observed during early flower development (before stage 9) than during late developmental stages (after stage 12) (Fig. 4l and Supplementary Figs. 7e, 8d), which further revealed possible similar and overlapping functions of FYF and AGL19/AGL14/SOC1. GUS staining was detected in sepal/petal organs and was absent in the AZ of AGL19::GUS (Fig. 4m, n) and SOC1::GUS flowers ( Supplementary Fig. 8e-g), suggesting that AGL19/14 and SOC1 might have part of the functions of FYF in regulating flower senescence but not abscission. Although SOC1 and AGL19 might also be involved in regulating flower senescence, they have very low or complete FYF activates FYL1 and AGL19/14/SOC1 expression to enhance the regulation of flower abscission and senescence. Since FYF has the same function as FYL1 and AGL19/14/SOC1 to regulate abscission and senescence of the sepal/petal, respectively, we were also interested in determining how they work together. When the expression pattern of endogenous FYL1 and AGL19/ 14/SOC1 was analyzed in 35 S::FYF flowers, we found that the expression of all three genes was clearly upregulated (Fig. 4o). Our results revealed that FYL1 was activated by FYF in the AZ during flower development and could enhance the function of the FYF gene in suppressing sepal/petal abscission, whereas AGL19/14/SOC1 were activated by FYF in sepals/petals during flower development, which could enhance the function of the FYF gene in suppressing sepal/petal senescence. Interestingly, we found that FYF, FYL1, AGL14, and SOC1 expression was also upregulated in 35 S::AGL19 flowers (Fig. 4p). This result suggested that FYF and other FYF-like genes with the strong repressor role, such as AGL19, could reciprocally activate each other to enhance the suppression of sepal/petal senescence.
FYF activates FYL2 expression to regulate flower senescence possibly through a feedback loop. Exploring how FYF competes with FYL2 to oppositely regulate sepal/petal senescence is interesting. When the expression pattern of endogenous FYL2 was analyzed in 35S::FYF flowers, we found that FYL2 expression was upregulated (Fig. 4o). Our results revealed that FYL2 was activated by FYF during flower development and that FYL2 could possibly form a feedback loop to contend with endogenous FYF function and to more appropriately control flower senescence. This assumption was further supported by the downregulation of FYL2 expression in fyf/agl15 double mutants ( Supplementary  Fig. 9a). We also found that FYL2 expression was upregulated in 35S::AGL19 flowers (Fig. 4p). This result suggested that FYF and AGL19 might control sepal/petal senescence by regulating FYL2 in a similar way.

Discussion
The Arabidopsis MADS box gene FYF can regulate flower organ senescence and abscission 1 . Ectopic expression of FYF caused a delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis and Eustoma grandiflorum 1 . This study further showed that two tandem repeat FYF-like genes, FYL1, and FYL2, and three other FYF-like genes, AGL19/14 and SOC1, in Arabidopsis were also involved in the regulation of flower organ abscission and/or senescence, and their functions were complementary or antagonistic to FYF.
FYL1 was found to act as a repressor to suppress abscission in sepals/petals with a complementary function to FYF (Fig. 5a). Unexpectedly, FYL2 could function as an activator and antagonize FYF in promoting the senescence of sepals/petals (Fig. 5a). The functions of FYL1/2 are correlated with their expression pattern since FYL1 was specifically expressed in the AZ of sepals/petals, whereas FYL2 expression was detected in the organs of sepals/ petals. The amino acid identity and the phylogenetic tree relationship 6 revealed that FYL1/2 were possibly the result of two duplication events. The first event generated an FYL1/2 ancestor from FYF, and the second event produced the two tandem repeats FYL1 and FYL2 from this FYL1/2 ancestor. The conserved role of FYF/FYL1/FYL2 during evolution in regulating flower senescence and/or abscission was further supported by their ability to interact with the same MADS box proteins AGL6 and SEP1. The original FYF gene must have contained both regulatory elements in its promoter or introns 1,33,[41][42][43][44][45] , which are required for its expression in the organs and AZ of sepals/petals. In the AZ, FYF specifically interacts with AGL6 to suppress abscission of the sepals/petals (Fig. 5a, b). In the sepal/petal organs, FYF can interact with either AGL6 or SEP1 to suppress senescence (Fig. 5a, c). In the tandem repeat genes FYL1 and FYL2, the subfunctional alteration of the regulatory elements during evolution resulted in restriction of the expression of FYL1 in the AZ and FYL2 in sepal/petal organs. FYL1 should maintain the conserved ability of FYF to interact with AGL6 together to suppress the abscission of sepals/petals (Fig. 5a,  b). Conversely, FYL2 only retained the conserved ability of FYF to interact with AGL6 and SEP1 in regulating sepal/petal senescence (Fig. 5a, c). However, FYL2 evolved into a role antagonistic to FYF and possibly helped to control FYF activity through a feedback loop to protect the flower buds from senescence and ensure the final senescence of the mature flowers (Fig. 5a, c).
In addition to FYL1/2, three putative FYF-like genes in the SOC1 subgroup, AGL19, AGL14, and SOC1, which are closely Fig. 3 The distance-measuring system validated that Arabidopsis AGL15-AGL6 forms stable abscission tetrameric complexes with FYF-AGL6 and FYL1-AGL6. The steady state of dimerization of the protein complexes FYF-AGL6 (a), FYL1-AGL6 (b), AGL15-AGL6 (c), FYF-AGL15 (d), and FYL1-AGL15 (e) is revealed in scatter diagrams showing pFRET and FRET efficiency. The black dots show the independent cell nuclei, and the yellow boxes indicate the steady-state FRET efficiency range for the protein complex. The mean value of FRET efficiency in the steady state is shown at the top of the schematic model, which is the baseline. The protein fused with CFP/YFP was attached by blue/yellow spots. f Schematic model of the protein interactions in the Arabidopsis abscission complexes (1) FYF-AGL6-AGL6-AGL15 and (2) FYL1-AGL6-AGL6-AGL15. Scatter diagram of the raw FRET (pFRET) and FRET efficiency values of the dimer pairs in adjacent lines AGL6-FYF (g) and AGL6-AGL15 (h) and diagonal lines AGL6-AGL6 (i) and FYF-AGL15 (j) in the abscission complex FYF-AGL6-AGL6-AGL15, with a different number of cell nuclei measured. The green dotted lines indicate the overlapping distribution range at the steady state. The yellow boxes (in g, h, j) indicate the baselines obtained for the dimer pairs. The protein fused with CFP/YFP was attached by blue/yellow spots. k Schematic model and FRET efficiency of four different pairs (two adjacent lines and two diagonal lines) of the protein interactions in the Arabidopsis stable abscission complex FYF-AGL6-AGL6-AGL15. The two adjacent lines (black) show similar FRET efficiencies (31%/30%), and the two diagonal lines (blue) show similar FRET efficiencies (29%/24%). Scatter diagram of the raw FRET (pFRET) and FRET efficiency values of the dimer pairs in adjacent lines AGL6-FYL1 (l) and AGL6-AGL15 (m) and diagonal lines AGL6-AGL6 (n) and FYL1-AGL15 (o) in the abscission complex FYL1-AGL6-AGL6-AGL15, with a different number of cell nuclei measured. The green dotted lines indicate the overlapping distribution range at the steady state. The yellow boxes (in l, m, o) indicate the baselines obtained for the dimer pairs. The protein fused with CFP/YFP was attached by blue/yellow spots. p Schematic model and FRET efficiency of four different pairs (two adjacent lines and two diagonal lines) of the protein interactions in the Arabidopsis stable abscission complex FYL1-AGL6-AGL6-AGL15. The two adjacent lines (black) show similar FRET efficiencies (32%/32%), and the two diagonal lines (blue) show similar FRET efficiencies (36%/34%). related to FYF/FYL1/FYL2 genes based on the phylogenetic tree relationship 6,16 , were also characterized. Based on the results of the functional analysis and the expression patterns of these three genes, we found that AGL19, AGL14, and SOC1 were all involved in the regulation of senescence but not abscission of flower organs (Fig. 5a, c). AGL19 might play a stronger repressive role than AGL14/SOC1, which functions similarly to FYF in suppressing flower senescence (Fig. 5a). Although AGL19/14 and SOC1 were found to be involved in regulating flower senescence, similar to FYF/FYL2, they seemed to perform their function in a different way in terms of finding interacting partners to form functional complexes. For example, FYF/FYL1/FYL2 can interact sufficiently with AGL6/SEP1, whereas AGL19/14/SOC1 can not interact with AGL6/SEP1. This finding indicated that the AGL19/14/ SOC1 subgroup might have their own interacting partners in regulating flower senescence that differ from those of FYF/FYL1/ FYL2 during evolution.
One interesting finding is that these FYF-like genes could regulate the expression of each other. For example, FYF could positively regulate the expression of FYL1 and AGL19/14/SOC1 to enhance suppression of the abscission and senescence of flower organs, respectively, whereas AGL19 could positively regulate the expression of FYF and AGL14/SOC1 to enhance the suppression of flower organ senescence. This positive reciprocal regulatory network among the FYF-like genes should provide a mechanism to ensure the suppression of senescence/abscission during the early stage of flower organ development (Fig. 5b, c). In addition, we also found a possible feedback loop regulatory mechanism between FYF and its opposite functional activator FYL2. FYF could activate the expression of FYL2, which possibly sequentially antagonized the activity of FYF. In this case, FYF activity will be countervailed at an appropriate level by FYL2, which is high in early and low in late flower development and ensures that sepal/ petal senescence will occur after flower maturation and will not occur in the flower bud stage (Fig. 5c).
Our findings reveal the potential immense complexity of the different combinations of FYF-like, A/E functional, and AGL15/ 18-like proteins in forming heterotetrameric abscission/senescence complexes (Fig. 5d). This complicated gene redundancy might explain why it is difficult to identify the senescence/ abscission mutant phenotype in a single gene mutation for these genes. In an attempt to mutate FYF (key gene in FYF-like) and AGL15 (key gene in AGL15/18-like) simultaneously, T-DNA mutants for each gene were crossed to generate fyf/agl15 double mutations. Very interestingly, early senescence and abscission of the flowers was observed in these fyf/agl15 double mutants ( Supplementary Fig. 9). This result strongly supported our assumption that abscission/senescence heterotetrameric complexes are at least composed of different combinations of FYF-like and AGL15/18-like proteins. Simultaneous mutations in FYF and AGL15 proteins will disrupt the functions of various combinations of the complexes and result in early senescence/abscission mutant phenotypes. In conclusion, our findings not only greatly expand the current knowledge concerning the multifunctional evolution of FYF-like genes in regulating flower senescence/ abscission but also provide an excellent example for the study of diverse functionalizations of duplicate gene pairs in plants.

Methods
Plant materials and growth conditions. The T-DNA insertion mutants of FYF (fyf, SALK_047915) and AGL15 (agl15, SALK_076234C) mutants Arabidopsis seeds were obtained from the Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH, USA. Seeds for Arabidopsis were germinated and grown as described previously 1,2,46 . Arabidopsis seeds were sterilized and placed on agar plates containing 1/2 X Murashige & Skoog medium 47 at 4°C for 2 days. Before being transplanted to soil, the seedlings were grown in growth chambers under long-day conditions (16 h light/8 h dark) at 22°C for 10 days. The light intensity of the growth chambers was 150 μE m −2 s −1 .
Cloning of the cDNA for FYL1, FYL2, AGL6, AGL19, AGL14, SOC1, and AGL15 from Arabidopsis. For 35S::MADS constructs, the cDNAs for FYL1, FYL2, AGL6, AGL19, AGL14, SOC1, and AGL15 were obtained by PCR amplification using genespecific 5′ and 3′ primers. The primers contained the XbaI and KpnI recognition sites to facilitate the cloning of the cDNAs. The XbaI-KpnI fragment containing the cDNA was cloned into the binary vector pEpyon-22K 1 under the control of the :AGL19 (f, g), and 35S::AGL19+SRDX (h, i) plants after exposure to ethylene. Wild-type flowers were senescent (arrowed in d, e), whereas 35S::AGL19 and 35S::AGL19+SRDX flowers were not senescent/ abscised. Bars = 2 mm. Detection of EDF1-4 and ERF1 (j) and BOP1/2, IDA and HAESA (k) expression in 35S::AGL19 and 35S::AGL19+SRDX Arabidopsis. Error bars show ± SD. n = 3 biologically independent samples. The expression of each gene in the transgenic plants is given relative to that of the wild-type plant, which was set at 1. The letter "a", "b" and "c" indicates significant difference from the wild-type (WT) value (a: P < 0.05, b: P < 0.01, and c: P < 0.001). The two-sided Student's t-test was used. l Detection of AGL19 expression before (BP) and after (AF) pollination. m, n GUS was stained in the sepals/petals of flowers of AGL19::GUS Arabidopsis. GUS was strongly stained in stage 8-10 young flower buds and gradually decreased in the mature flowers during the late stage (after stage 12) of flower development. The numbers indicate the different developmental stages of Arabidopsis flowers. n is the magnified view from (m). s: sepal, p: petal, st: stamen. Bars = 1 mm. Detection of FYL1/FYL2/AGL19/AGL14/SOC1 expression in 35S::FYF Arabidopsis (o) and FYF/FYL1/ FYL2/AGL14/SOC1 expression in 35S::AGL19 Arabidopsis (p). Error bars show ± SD. n = 3 biologically independent samples. The expression of each gene in the transgenic plants is given relative to that of the wild-type plant, which was set at 1. The letter "a", "b" and "c" indicates significant difference from the wild-type (WT) value (a: P < 0.05, b: P < 0.01, and c: P < 0.001). The two-sided Student's t-test was used.
CaMV 35S promoter and used for plant transformation. Sequences for the primers are listed in the Supplementary Table 1.
Cloning of the promoter DNA fragment from Arabidopsis. For the FYL1::GUS and FYL2::GUS constructs, the promoter regions which included the 5′UTR and first intron for FYL1 (2.65 kb) and FYL2 (2.43 kb) were obtained by PCR amplification using specific primer pairs from the genomic DNA followed by cloning into the pGEM-T easy vector (Promega, Madison, WI, USA). These promoter fragments were then subcloned into the linker region before the β-Glucuronidase (GUS) coding region in the binary vector pEpyon01k 1,2 . For the AGL6::GUS, AGL15::GUS, AGL19::GUS, and SOC1::GUS constructs, the promoter regions which included the 5'UTR and first intron for AGL6 (4.96 kb), AGL15 (0.94 kb), AGL19 (4.59 kb) and SOC1 (5.68 kb) were obtained by PCR amplification and these promoter fragments were subcloned into the linker region before the β-Glucuronidase (GUS) coding region in the binary vector pEpyon01k in the same manner as FYL1/2::GUS. Sequences for the primers are listed in Supplementary  Table 1. For the IDA::FYL1 construct, the IDA promoter (1.43 kb) was obtained by PCR amplification as described previously 1 . The cDNA for FYL1 was obtained by PCR amplification. The IDA promoter and the cDNA for FYL1 were subcloned into the modified binary vector pEpyon-12K 1 . Sequences for the primers are listed in Supplementary Table 1.
Construction of the MADS + VP16 constructs. For the 35S::FYL1+VP16, and 35S::FYL2+VP16 construct, the cDNAs for FYL1/FYL2 were obtained by PCR amplification and cloned into the pEpyon-2bK plasmid upstream of the VP16-AD fragment sequence, under the control of the CaMV 35S promoter as described previously 1,2 . The sequences for the primers were listed in Supplementary Table 1.  5 The functional evolution and regulatory network of the FYF-like genes in regulating flower senescence/abscission. a In Arabidopsis, six FYF-like genes in two subgroups (FYF/FYL1/FYL2 and AGL19/AGL14/SOC1) were all involved in regulating flower senescence and/or abscission. In the FYF subgroup, FYF acts as a repressor (R) in suppressing both flower senescence (indicated by a blue box) and abscission (indicated by a pink box), and FYL1 acts as a repressor (R) and only suppresses flower abscission (indicated by a pink box). FYL2 functions as an activator (A) in promoting flower senescence (indicated by a gray box). In the SOC1 subgroup, AGL19, AGL14, and SOC1 function as repressors (R) and have only one function in suppressing flower senescence (indicated by a light blue box), with the effect of AGL19 being stronger than that of AGL14/SOC1. In addition, the A/E functional genes AGL6/ SEP1 and AGL15/18-like gene AGL15 (as a repressor) can regulate senescence (indicated by a blue box) by interacting with FYF/FYL2, whereas AGL6 and AGL15 can also regulate abscission (indicated by a pink box) by interacting with FYF/FYL1. The size of the letter R in the box correlated with the strength of the repressor for the MADS box proteins. b In the AZ of the perianth, FYF and FYL1 complement each other by forming two identified heterotetrameric abscission complexes, FYF/AGL6/AGL6/AGL15 and FYL1/AGL6/AGL6/AGL15, suppressing flower abscission through the downregulation (⊣) of BOP1/2 and IDA/HAESA expression. c In sepals/petals, FYF, AGL19, AGL14, and SOC1 functioned antagonistically to FYL2 in suppressing flower senescence. An identified FYF/AGL6/AGL6/AGL15 together with FYF/SEP1/Y, AGL19/X/Y, AGL14/X/Y, and SOC1/X/Y heterotetrameric senescence complexes suppressed sepal/petal senescence through the downregulation (⊣) of ethylene downstream gene expression. In contrast, FYL2/AGL6/Y and FYL2/SEP1/Y heterotetrameric complexes promoted sepal/petal senescence by the activation (→) of ethylene downstream gene expression, possibly through a negative feedback loop to FYF/X/Y, AGL19/X/Y, AGL14/X/Y, and SOC1/X/Y. d In these cases from (c), the heterotetrameric complexes are composed of FYF-like, X (in red) and Y (in blue) proteins. FYF-like can be either one of the FYF/FYL1/FYL2/AGL19/AGL14/SOC1, X can be AGL6, SEP1, or any unidentified A/E proteins, whereas Y can be AGL15, AGL18, or any unidentified AGL15/18-like proteins.
Plant transformation and transgenic plant analysis. A floral dip method as described elsewhere 48 was used to introduce constructs made in this study in the Agrobacterium tumefaciens strain GV3101 into Arabidopsis plants. PCR and RT-PCR analyses were used to verified the transformants that survived in medium containing kanamycin (50 µg/ml). To generate 35S::FYF/35S::FYL2 Arabidopsis, constructs of 35S::FYL2 which contained hygromycin resistant gene were cotransformed with 35S::FYF (kanamycin resistant) into Arabidopsis plants. Transformants that survived in medium containing both kanamycin (50 µg/ml) and hygromycin (5 µg/ml) were selected for further analysis. To generate fyf/agl15 double mutant Arabidopsis, homozygous fyf were crossed with the agl15 T-DNA mutants in the Columbia background and F 1 plants were used to further generate the F 2 generation. One quarter of the F 2 plants were fyf/agl15 and were further verified and selected for further analysis.